Epidermal growth factor receptor (EGFR)-targeted therapy in combination with radiation has been demonstrated to improve locoregional control and survival of patients with head and neck squamous cell carcinoma (HNSCC) in a randomized phase III clinical trial. Based on the encouraging pre-clinical studies of dual EGFR/vascular endothelial growth factor receptor (VEGFR) inhibition explored during the first five years of this SPORE, we are now investigating EGFR-VEGFR co-targeting using ZD6474 and radiation in the same clinical setting. Although this approach carries an enormous therapeutic potential, it is very likely that resistance to inhibition of these pathways will emerge as a potential obstacle to be overcome in clinical practice. The main goal of this Project 3 is to identify the potential pathways of resistance to ZD6474 employing high throughput methodology, consisting of tissue proteomics (reverse phase protein arrays [RPPA], and phosphorylated receptor tyrosine kinase arrays [p-RTK]) and cytokine and angiogenic factors (CAF) profiling. We will apply these three methods in both the pre-clinical (in vitro and in vivo models) and clinical settings (archival and prospectively collected samples from an ongoing trial of ZD6474 plus chemoradiation in patients with locoregionally advanced HNSCC). Aim 1 will determine the sensitivity of HNSCC cell lines to ZD6474 and identify signatures of in vitro resistance to the drug. In Aim 2 we will determine in vivo resistance signatures to the drug using orthotopic xenograft models subjected to treatment with ZD6474 +/- radiation. In Aim 3, we will identify signatures predictive of poor outcome in archival samples obtained from 3 cohorts of patients and develop a predictive model combining molecular signature information from pre-clinical aims and archival specimens. This overall predictive model will be prospectively evaluated in the context of the ongoing study of ZD6474 plus chemoradiation. This strategy will allow us to: 1) identify potential pathways of resistance to be targeted in future (pre)clinical studies, 2) validate pre-clinical models for identifying resistance to targeted agents and 3) develop high-throughput technology-based predictive models that will reflect both prognosis and resistance to dual EGFR and VEGFR inhibition and can inform the design of future therapeutic strategies.